1. Analysis of RPGR Isoforms Mutations in the gene for Retinitis Pigmentosa GTPase Regulator (RPGR) account for disease in up to 20% of all retinitis pigmentosa (RP) patients and are therefore the most frequent cause of RP. Functional and pathogenic studies of RPGR have been hampered by its complex expression pattern and by technical difficulties arisen from its highly repetitive sequences. The precise coding sequence of RPGR is not known and there have been conflicting reports about the subcellular localization of RPGR isoforms and its interacting protein RPGRIP1 in the retina. We have been performing experiments to clarify these two issues, which are important for the design of replacement gene therapies. 2. Replacement gene therapy In our studies in mice, we have found that RPGR-interacting protein 1 (RPGRIP1) is localized primarily in the photoreceptor connecting cilium where it anchors the RPGR (retinitis pigmentosa GTPase regulator) protein, and its function is essential for photoreceptor maintenance. Genetic defect in RPGRIP1 is a known cause of Leber congenital amaurosis (LCA), a severe, early-onset form of retinal degeneration. We evaluated the efficacy of replacement gene therapy in a murine model of LCA carrying a targeted disruption of RPGRIP1. The replacement construct, packaged in an AAV8 vector, utilized a rhodopsin kinase (RK) gene promoter to drive RPGRIP1 expression. Both promoter and transgene were of human origin. Following subretinal delivery of the replacement gene in the mutant mice, human RPGRIP1 was expressed specifically in photoreceptors, localized correctly in the connecting cilia, and it restored the normal localization of RPGR. Electroretinogram and histologic examinations showed better preservation of rod and cone photoreceptor function and improved survival in the treated eyes. This study demonstrates the efficacy of human gene replacement therapy and validates a gene therapy design for future clinical trials in patients afflicted with this condition. Our results also have therapeutic implications for other forms of retinal degenerations attributable to a ciliary defect. In collaboration with Dr. Michael Sandberg at the Harvard Medical School/Mass. Eye &Ear Infirmary, we are planning on performing follow up experiments to evaluate the therapeutic potential of this approach for clinical applications. 3. Usher syndrome is a genetic disorder affecting both vision and hearing. It is classified into three clinical types. Among them, type II (USH2) is the predominant form accounting for about 70% of all Usher syndrome cases. Three genes, USH2A, USH2C and USH2D, underlie the development of USH2, and they encode usherin, Very Large G protein-coupled Receptor-1 (VLGR1) and whirlin, respectively. In collaboration with Dr. Jun Yang (University of Utah), we have recently shown that the long whirlin isoform organizes the formation of a multi-protein complex in vivo that includes usherin and VLGR1. Targeted disruption of whirlin near its N-terminus in mice, which eliminates the long isoform, abolishes the normal subcellular localization of the two partner USH2 proteins, causes structural defects in photoreceptors and inner ear hair cells, and leads to death of these cells over time. We present the first definitive evidence that the USH2 proteins mark the boundary of the periciliary membrane complex, which was first described in frog photoreceptors and is thought to play a role in regulating protein transport through the connecting cilia. Furthermore, defects in all USH2 proteins share a common pathogenic pathway by disrupting the periciliary membrane complex in photoreceptors. These data represent a major step forward in our understanding of the Usher 2 disease process. We are currently performing further studies on a number of USH1 genes and new, putative mouse models of human Usher syndrome.